Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1α: effect on α2A-adrenoceptor-Gi1α fusion protein activation

Abstract

Fusion proteins were constructed between the porcine α_2A‐adrenoceptor and either wild‐type (Cys³⁵¹) or a pertussis toxin‐resistant (Gly³⁵¹) form of the G protein G_i1α. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration‐dependent stimulation of their high affinity GTPase activity. The α_2A‐adrenoceptor‐wild type G_i1α fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly³⁵¹ G_i1α. Treatment of the fusion proteins as agonist‐regulated enzymes allowed measurement of V _max and turnover number for adrenaline‐stimulation of the GTPase activity of each fusion construct. The turnover number of the α_2A‐adrenoceptor‐Cys³⁵¹Gly G_i1α fusion protein was only 44% of that for the α_2A‐adrenoceptor‐wild type G_i1α fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist‐occupied receptor to activate the mutant.